1. Field of the Invention
The present invention provides isolated human heparanase polypeptides, and the isolated polynucleotide molecules that encode them, as well as vectors and host cells comprising such polynucleotide molecules. The invention also provides a method for the identification of an agent that alters heparanase activity.
2. Related Art
Heparanase is a human enzyme that can degrade both heparin proteoglycans (HPG) and heparan sulfate proteoglycans (HSPG). Heparanase activity in mammalian cells is well known. The activity has been identified in various melanoma cells (Nakajima, et al., Cancer Letters 31: 277-283, 1986), mammary adenocarcinoma cells (Parish, et al., Int. J. Cancer, 40: 511-518, 1987), leukemic cells (Yahalom, et al., Leukemia Research 12: 711-717, 1988), prostate carcinoma cells (Kosir, et al., J. Surg. Res. 67: 98-105, 1997), mast cells (Ogren and Lindahl, J. Biol. Chem. 250: 2690-2697, 1975), macrophages (Savion, et al., J. Cell. Physiol, 130: 85-92, 1987), mononuclear cells (Sewell, et al., Biochem. J. 264: 777-783, 1989), neutrophils (Matzner, et al. 51: 519-524, 1992, T-cells (Vettel et al., Eur J. Immunol. 21: 2247-2251, 1991), platelets (Haimovitz-Friedman, et al., Blood 78: 789-796, 1991), endothelial cells (Godder, et al., J. Cell Physiol. 148: 274-280, 1991), and placenta (Klein and von Figura, BBRC 73: 569, 1976). An earlier report that human platelet heparanase is a member of the CXC chemokine family (Hoogewerf et al., J.Biol. Chem. 270: 3268-3277) is in error.
Elevated heparanase activity has been documented in mobile, invasive cells. Examples include invasive melanoma, lymphoma, mastocytoma, mammary adenocarcinoma, leukemia, and rheumatoid fibroblasts. Heparanase activity has also been documented in non-pathologic situations involving the migration of lymphocytes, neutrophils, macrophages, eosinophils and platelets (Vlodavsky et al., Invasion Metastasis 12: 112-127, 1992).
A number of uses have been proposed for bacterial heparanases. One such use is described in Freed et al. (Ann. Biomed. Eng. 21: 67-76 (1993)), wherein purified bacterial heparanase is immobilized onto filters and connected to extracorporeal devices for use in the degradation of heparin and the neutralization of its anticoagulant properties post surgery.
Other proposed uses for bacterial heparanases include the use of heparanase in a method for inhibiting angiogenesis (U.S. Pat. No. 5,567,417), an application of the enzyme as a means of decreasing inflammatory responses (WO 97/11684), and the use of heparanase-inhibiting compositions for preventing tumor metastasis (U.S. Pat. No. 4,882,318).
In view of the observation that heparanase activity is present in mobile, invasive cells associated with pathologic states, it may be hypothesized that an inhibitor of heparanase would broadly influence the invasive potential of these diverse cells. Further, inhibition of heparan sulfate degradation would inhibit the release of bound growth factors and other biologic response modifiers that would, if released, fuel the growth of adjacent tissues and provide a supportive environment for cell growth (Rapraeger et al., Science 252: 1705-1708, 11991). Inhibitors of heparanase activity would also be of value in the treatment of arthritis, asthma, and other inflammatory diseases, vascular restenosis, atherosclerosis, tumor growth and progression, and fibro-proliferative disorders.
A major obstacle to designing a screening assay for the identification of inhibitors of mammalian heparanase activity has been the difficulty of purifying any mammalian heparanase to homogeneity so as to determine its structure, including its amino acid sequence. For this reason, therapeutic applications of mammalian heparanase, or of inhibitors of mammalian heparanase, have been based on research carried out using bacterial heparanase.
WO 91/02977 describes a substantially, but partially, purified heparanase produced by cation exchange resin chromatography and the affinity absorbent purification of heparanase-containing extract from the human SK-HEP-1 cell line. WO 91/02977 also describes a method of promoting wound healing utilizing compositions comprising a xe2x80x9cpurifiedxe2x80x9d form of heparanase. This enzyme was not thoroughly characterized, and its amino acid sequence was not determined. WO 98/03638 describes a method for the pourification of mammalian heparanase from a heparanase-containing material, such as human platelets. However, the amino acid sequence of this heparanase, and the sequence of the polynucleotide molecule that encodes it, are not disclosed in this reference. Furthermore, this heparanase is characterized only as having a native molecular mass of about 50 kDa, and as degrading both heparin and heparan sulfate.
Although a number of assays for heparanase have been described, the complexity of the HSPG substrate has caused methods for assay of heparanase activity to be rudimentary and lacking in kinetic sophistication. Haimovitz-Friedman et al. (Blood 78: 789-796, 1991) describe an assay for heparanase activity that involves the culturing of endothelial cells in radiolabeled 35SO4 to produce radiolabeled heparan sulfate proteoglycans, the removal of the cells which leaves the deposited extracellular matrix that contains the 35S-HSPG, the addition of potential sources of heparanase activity, and the detection of possible activity by passing the supernatant from the radiolabeled extracellular matrix over a gel filtration column and monitoring for changes of the size of the radiolabeled material that would indicate that HSPG degradation had taken place. However, this assay cannot be used in a high-throughput screening format.
Nakajima et al. (Anal. Biochem. 196: 162-171, 1986) describe a solid-phase substrate for the assay of melanoma heparanase activity. Heparan sulfate from bovine lung is chemically radiolabeled by reacting it with [14C]-acetic anhydride. Free amino groups of the [14C]-heparan sulfate were acetylated and the reducing termini were aminated. The [14C]-heparan sulfate was chemically coupled to an agarose support via the introduced amine groups on the reducing termini. However, the usefulness of the Nakajima et al. assay is limited by the fact that the substrate is an extensively chemically modified form of naturally occurring heparan sulfate.
Khan and Newman (Anal. Biochem. 196: 373-376, 1991) describe an indirect assay for heparanase activity. In this assay, heparin is quantitated by its ability to interfere with the color development between a protein and the dye Coomassie brilliant blue. Heparanase activity is detected by the loss of this interference. This assay is limited in use for screening because it is so indirect that other non-heparin compounds could also interfere with the protein-dye reaction.
In view of the foregoing, it will be clear that there is a need in the art for recombinantly produced human heparanase.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding human heparanase polypeptides. Unless otherwise indicated, any reference herein to a xe2x80x9chuman heparanase polypeptidexe2x80x9d will be understood to encompass human pre-pro-heparanase, pro-heparanase, and both the 8 kDa and the 56 kDa subunits of the human heparanase enzyme. Pre-pro-heparanase refers to an amino acid sequence which includes a leader sequence, and which can be processed to remove 48 amino acids yielding both the 8 kDa and the 56 kDa subunits of the human heparanase enzyme; pro-heparanase refers to the enzymatically inactive, full-length molecule from which the signal peptide has been removed and which can be processed to yield both the 8 kDa and the 56 kDa subunits of the human heparanase enzyme. Fragments of human heparanase polypeptides are also provided. Unless otherwise indicated, any reference herein to a xe2x80x9chuman heparanase enzymexe2x80x9d will be understood to refer to a non-covalently associated complex of the 56 kDa and the 8 kDa human heparanase polypeptides.
In a preferred embodiment, the nucleic acid molecules comprise an isolated polynucleotide having a nucleotide sequence encoding a human heparanase polypeptide selected from the group consisting of: a human pre-pro-heparanase polypeptide having the complete amino acid sequence of SEQ ID NO:2; a human pro-heparanase polypeptide having the amino acid sequence at residues 23 through 530 of SEQ ID NO:2; the 8 kDa subunit of human heparanase having the amino acid sequence at residues 23 through 96 of SEQ ID NO:2; and the 56 kDa subunit of human heparanase having the amino acid sequence at residues 145 through 530 of SEQ ID NO:2.
In another preferred embodiment, the nucleic acid molecules comprise a polynucleotidc having a nucleotide sequence selected from the group consisting of the complete nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence at residues 67 through 1590 of SEQ ID NO: 1, the nucleotide sequence at residues 433 through 1590 of SEQ ID NO:1, and the nucleotide sequence at residues 67 through 288 of SEQ ID NO:1. In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide encoding a human heparanase polypeptide, or fragments thereof.
The present invention also provides vectors comprising the isolated nucleic acid molecules of the invention, host cells into which such vectors have been introduced, and recombinant methods of obtaining a human heparanase polypeptide comprising culturing the above-described host cell and isolating the human heparanase polypeptides.
In another aspect, the invention provides isolated human heparanase polypeptides, as well as fragments thereof. In a preferred embodiment, the human heparanase polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of a human prc-pro-heparanase having the complete amino acid sequence of SEQ ID NO:2, an amino acid sequence of a human pro-heparanase having the amino acid sequence at residues 23 through 530 of SEQ ID NO:2, an amino acid sequence of the 8 kDa subunit of human heparanase having amino acid sequence at residues 23 through 96 of SEQ ID NO:2, and an amino acid sequence of the 56 kDa subunit of human heparanase having the amino acid sequence at residues 145 through 530 of SEQ ID NO:2.
In a preferred embodiment, the human heparanase polypeptides of the invention are expressed from an isolated nucleic acid molecule encoding a polypeptide selected from the group consisting of a human pre-pro-heparanase polypeptide having the complete amino acid sequence of SEQ ID NO:2; a human pro-heparanase polypeptide having the amino acid sequence at residues 23 through 530 of SEQ ID NO:2; the 8 kDa subunit of human heparanase having the amino acid sequence at residues 23 through 96 of SEQ ID NO:2; and the 56 kDa subunit of human heparanase having the amino acid sequence at residues 145 through 530 of SEQ ID NO:2.
In another preferred embodiment, the human heparanase polypeptides of the invention are expressed from an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: the complete nucleotide sequence of SEQ ID NO: 1; the nucleotide sequence at residues 67 through 1590 of SEQ ID NO:1; the nucleotide sequence at residues 433 through 1590 of SEQ ID NO:1; and the nucleotide sequence at residues 67 through 288 of SEQ ID NO: 1. Isolated antibodies, both polyclonal and monoclonal, that bind specifically to human heparanase polypeptides are also provided.
The invention also provides a human heparanase enzyme comprising an isolated human heparanase polypeptide comprising the amino acid sequence at residues 145 through 530 of SEQ ID NO:2 and an isolated human heparanase polypeptide comprising the amino acid sequence at residues 23 through 96 of SEQ ID NO:2.
The invention also provides a method for the identification of an agent that alters heparanase activity, said method comprising:
(a) determining the activity of any of the above-described human heparanase enzyme
(i) in the presence of a test agent; and
(ii) in the absence of said test agent; and
(b) comparing the heparanase activity determined in step (a)(i) to the heparanase activity determined in step (a)(ii); whereby a change in heparanase activity in sample (a)(i) has compared to sample (a)(ii) indicates that said agent alters the activity of said human heparanase.